316 CHANGES IN MPF ACTIVITY IN PORCINE OOCYTES FOLLOWING ACTIVATION BY VARIOUS METHODS

Abstract

In mammalian oocytes metaphase II arrest is maintained by high activity of the M-phase promoting factor (MPF), which is a heterodimer of cdc2 and cyclin B. The repetitive increase in the intracellular free calcium concentration at fertilization leads to the inactivation of MPF; low MPF activity will then stimulate the resumption of meiosis and entry into interphase. Most oocyte activation methods are able to trigger only a single calcium transient that may not be sufficient to keep MPF levels low enough to induce pronuclear formation. The objective of this study was to determine the profile of MPF activity after different oocyte activation protocols. Mature pig oocytes from prepubertal gilts were allocated into three groups: (1) oocytes activated by two consecutive electrical pulses; (2) oocytes electroporated and incubated for 4 h in 100 μM butyrolactone I, an inhibitor of cdc2; (3) oocytes electroporated and treated for 5 h with 10 μg/mL cycloheximide, a protein synthesis blocker. Some oocytes were removed from the medium at 2, 4, and 6 h after the electrical pulse and used to determine MPF activity using a cdk1/cdc2 kinase assay kit. The remaining oocytes were placed into PZM-1 medium and cultured for 7 days. Data were compared using ANOVA. After the electrical stimulus the initial activity of MPF (0.25 pmol/min/oocyte) dropped in all cases, i.e., the amount of phosphate incorporated into histones decreased. However, in oocytes activated with an electrical pulse only, MPF activity that by had 4 h dropped to 0.15 pmol/min/oocyte started to increase and by 6 h it reached 0.2 pmol/min/oocyte. In the cycloheximide-treated oocytes this increase was negligible (by 6 h the activity was 0.12 pmol/min/oocyte) whereas incubation in butyrolactone resulted in a continuous decrease in MPF activity which by 6 h was as low as 0.07 pmol/min/oocyte. Developmental data of the activated oocytes are presented in Table 1. The percent of embryos cleaved and the percent of oocytes that formed blastocysts were higher in the cycloheximide-treated group than in controls. In contrast, the total cell number per blastocyst was the highest in oocytes that were activated by electroporation only. Inhibiting cdc2 kinase or protein synthesis helps to maintain low MPF activity after stimulation and results in a high cleavage ratio and blastocyst formation in the pig. Such treatment, however, had a negative effect on total cell number at the blastocyst stage.

Table 1. Developmental data of activated oocytes